Accepted Manuscript Title: Localisation of oxysterols at the sub-cellular level and in biological fluids Authors: Irundika HK Dias, Khushboo Borah, Berivan Amin, Helen R Griffiths, Khouloud Sassi, Gerard´ Lizard, Ane Iriondo, Pablo Martinez-Lage PII: S0960-0760(19)30173-6 DOI: https://doi.org/10.1016/j.jsbmb.2019.105426 Article Number: 105426 Reference: SBMB 105426 To appear in: Journal of Steroid Biochemistry & Molecular Biology Received date: 21 March 2019 Revised date: 25 June 2019 Accepted date: 9 July 2019 Please cite this article as: Dias IH, Borah K, Amin B, R Griffiths H, Sassi K, Lizard G, Iriondo A, Martinez-Lage P, Localisation of oxysterols at the sub-cellular level and in biological fluids, Journal of Steroid Biochemistry and Molecular Biology (2019), https://doi.org/10.1016/j.jsbmb.2019.105426 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Localisation of oxysterols at the sub-cellular level and in biological fluids Irundika HK Dias1*, Khushboo Borah2, Berivan Amin1, Helen R Griffiths1,2, Khouloud Sassi 3, 4, Gérard Lizard3, Ane Iriondo5, Pablo Martinez-Lage5 1. Aston Medical Research Institute, Aston Medical School, Aston University, Birmingham, B4 7ET, UK 2. Faculty of Health and Medical Sciences, University of Surrey, Stag Hill, Guildford, GU2 7XH, UK 3. Team Bio-PeroxIL, Biochemistry of the Peroxisome, Inflammation and Lipid Metabolism (EA7270)/University Bourgogne Franche-Comté/Inserm, 21000 Dijon, France 4. Univ. Tunis El Manar, Laboratory of Onco-Hematology (LR05ES05), Faculty of Medicine, Tunis, Tunisia 5. Department of Neurology, Center for Research and Advanced Therapies, CITA-Alzheimer Foundation, San Sebastian, Spain * Corresponding author Highlights Oxysterols are involved in many biological processes including the regulation of cholesterol homeostasis The location of oxysterol metabolising enzymes may play an important role in the oxysterol concentration in cellular micro-environments. Oxysterols in biological fluids can use as markers to analyse endogenous flaws in cholesterol/oxysterol homeostasis. ListACCEPTED of Abbreviations MANUSCRIPT 20-OHC 20‑hydroxycholesterol 22-OHC 22‑hydroxycholesterol 24-OHC 24‑hydroxycholesterol 1 25-OHC 25‑hydroxycholesterol 26-OHC 26‑hydroxycholesterol 27-OHC 27-hydroxycholesterol 5, 6-epox 5, 6-epoxy cholesterols 5α, 6α-epox 5α, 6α- epoxy cholesterol 5β, 6β-epox 5β, 6β- epoxy cholesterol 7-KC 7-ketocholesterol 7-K,25-OHC 7-keto-27-hydroxycholesterol 7α-OHC 7α-hydroxycholesterol 7α,25-OHC 7α, 25-dihydroxycholesterol 7α,27-OHC 7α, 27-hydroxycholesterol 7β-OHC 7β-hydroxycholesterol ABCA1 ATP-binding cassette transporter A1 ABCG1 ATP-binding cassette transporter G1 ACAT Acyl-CoA:cholesterol acyl transferase AD Alzheimer´s disease APP Amyloid precursor protein BBB Blood-brain barrier CH25H Cholesterol 25-hydroxylase ChEH Cholesterol-5, 6-epoxide hydrolase CNBP Cellular nucleic acid binding protein CYP46A1 Cholesterol 24-hydroxylase CYP46A1 Cytochrome P450 46A1 CYP7A1 Sterol 7α-hydroxylase CYP27 Sterol 27-hydroxylase CYP8B1 Cholesterol 12α‐hydroxylase DDA Dendrogenin A DHCAACCEPTED Dihydroxycholestanoic acid MANUSCRIPT ER Endoplasmic reticulum HSD Hydroxysteroid dehydrogenase HSD3B7 3β-hydroxysteroid dehydrogenase type 7 LDL Low density lipoproteins 2 LXR Liver X receptor MS Multiple Sclerosis NAFLD Non-alcoholic fatty liver disease OSBP Oxysterol binding protein PD Parkinson´s Disease POPC 1-palmitoyl-2-oleoyl-phosphatidylcholine SCAP SREBP cleavage activation protein SMO Smoothed receptor SREBP Sterol regulatory element binding protein StAR Steroidogenic acute regulatory protein THCA Trihydroxycholestanoic acid TSPO Mitochondrial translocator protein ROS Reactive oxygen species VLCFA Very long chain fatty acids WMH White Matter Hyperintensities Abstract Oxysterols are oxidized derivatives of cholesterol that are formed enzymatically or via reactive oxygen species or both. Cholesterol or oxysterols ingested as food are absorbed and packed into lipoproteins that are taken up by hepatic cells. Within hepatic cells, excess cholesterol is metabolised to form bile acids. The endoplasmic reticulum acts as the main organelle in the bile acid synthesis pathway. Metabolised sterols originating from this pathway are distributed within other organelles and in the cell membrane. The alterations to membrane oxysterol:sterol ratio affects the integrity of the cell membrane. The presence of oxysterols changes membrane fluidity and receptor orientation. It is well documented that hydroxylase enzymes located in mitochondria facilitate oxysterol productionACCEPTED via an acidic pathway. More recently, MANUSCRIPT the presence of oxysterols was also reported in lysosomes. Peroxisomal deficiencies favour intracellular oxysterols accumulation. Despite the low abundance of oxysterols compared to cholesterol, the biological actions of oxysterols are numerous and important. Oxysterol levels are implicated in the pathogenesis of multiple diseases ranging from 3 chronic inflammatory diseases (atherosclerosis, Alzheimer’s disease and bowel disease), cancer and numerous neurodegenerative diseases. In this article, we review the distribution of oxysterols in sub-cellular organelles and in biological fluids. Key words: Oxysterols; subcellular; membrane; mitochondria; peroxisomes; fluids 1-Biosynthesis of oxysterols in cells Oxysterols, oxygenated derivatives of cholesterols, are involved in many biological processes including the regulation of cholesterol homeostasis. While physiological levels of oxysterols are found to be implicated in many biological functions, imbalance of oxysterol homeostasis is reported to have impact on pathophysiology. Oxysterols are implicated as the initiation and progression of many chronic diseases in the literature; osteoporosis, age related macular degeneration, cataract, atherosclerosis, neurodegenerative diseases (e.g. Alzheimer’s and Parkinson’s diseases, and multiple sclerosis) [1-4]. Chemically, the addition of one or more oxygenated functional groups to 27-carbon cholesterol molecule changes its behaviour and receptor recognition. Oxidation can occur on the cluster of four hydrocarbon rings (A, B, C and D) or on the side chain of the cholesterol molecule. The double bond present on the B ring is the most energetically favourable target of free radical attack and therefore carbon atoms at the positions of 4, 5, 6 and 7 are more susceptible to free radical oxidation. Commonly observed B-ring oxidised oxysterols include hydroxylated compounds [7α- and 7β-hydroxycholesterol (7α-OHC and 7β-OHC)], oxysterols with a ketone group [7-ketocholesterol (7- KC)], epoxy cholesterols [5β, 6β- epoxy cholesterol (5β, 6β-epox), 5α, 6α- epoxy cholesterol (5α, 6α- epox)] and cholestan-3β, 5α, 6β‑triol [5-8]. In addition to ring oxidation, cholesterol undergoes side chain oxidation to generate oxysterols; 20‑hydroxycholesterol (20-OHC), 22‑hydroxycholesterol (22-OHC), 24‑hydroxycholesterol (24- OHC), 25-hydroxycholesterol (25-OHC) and 27-hydroxycholesterol (27-OHC, formally known as is (25R)ACCEPTED 26-hydroxycholesterol [9]). Further oxidation MANUSCRIPT on cholesterol molecules generates oxysterol molecules with two or more different functional groups (e.g. 7α, 25-dihydroxycholesterol, 7α, 27- hydroxycholesterol and 7-keto-27-hydroxycholesterol). The addition of a hydroxyl group to a side chain mainly occurs via enzymatic pathways that mainly regulate cholesterol homeostasis. Two main groups of enzymes; oxidoreductases and transferases, are known to participate in the metabolism of 4 oxysterols either through neutral or acidic pathways [10]. Most of the oxidoreductases are members of the heme-containing cytochrome P450 (CYP) family of enzymes (Table 1). Among them, cholesterol 7α-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27) were the first to be identified [11]. CYP7A1 is known to initiate the neutral or classical pathway and CYP27 is known to initiate the acidic or alternate pathway of oxysterol metabolism. Griffiths and Wang explained that once 7α position of cholesterol is hydroxylated, oxysterols become substrates for the enzyme HSD3B7 (3β-hydroxysteroid dehydrogenase type 7) and can be converted from their 3β-hydroxy-5- ene to 3-oxo-4-ene forms [12]. Another dehydrogenase enzyme, 11 β-dehydrogenase isoenzyme 1 (11βHSD1) is responsible for the reversible interconversion of 7-KC and 7β-OHC [13]. Non-heme iron-containing oxidoreductase, cholesterol 25-hydroxylase (CH25H), has recently received attention due to the involvement of its product, 25-OHC, in immunity control [14]. However, 25-OHC has been reported to be produced through other cytochromes (CYP3A4, CYP27, and CYP46A1) and also through free-radical reactions [15-17]. Substrate Enzyme Oxysterol derivative Cholesterol CYP3A4 4β-OHC Cholesterol CYP7A1 7α-OHC Cholesterol CYP11A1 20R-OHC Cholesterol CYP11A1 22R-OHC Cholesterol CYP46A1 24S-OHC Cholesterol CH25H 25-OHC Cholesterol CYP27 27-OHC 7α-OHC CYP27 7α,26-diOHC 22R-OHC CYP11A1 20R,22R-diOHC 24S-OHC CYP39A1 7α,24S-diOHC 25-OHC CYP7B1 7α,25-diOHC 27-OHC CYP7B1 7α,27-diOHC Table 1: Oxidoreductase enzymes involved in the metabolism of oxysterols Similar